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Biogeosciences, 5, 311–321, 2008 www.biogeosciences.net/5/311/2008/ Biogeosciences © Author(s) 2008. This work is distributed under the Creative Commons Attribution 3.0 License.

Distribution of micro-organisms along a transect in the South-East Pacific (BIOSOPE cruise) using epifluorescence microscopy

S. Masquelier and D. Vaulot Station Biologique de Roscoff, UMR 7144, CNRS et Universite´ Pierre et Marie Curie, Place G. Tessier, 29682, Roscoff, France Received: 6 July 2007 – Published in Biogeosciences Discuss.: 7 August 2007 Revised: 18 January 2008 – Accepted: 1 February 2008 – Published: 4 March 2008

Abstract. The distribution of selected groups of micro- 1 Introduction organisms was analyzed along a South-East Pacific Ocean transect sampled during the BIOSOPE cruise in 2004. The Unicellular picoplanktonic and less transect could be divided into four regions of contrasted than 2 µm in size (Sieburth et al., 1978) are found in marine trophic status: a High Nutrient Low (HNLC) at concentrations ranging from 102 to 105 and region (mesotrophic) near the equator, the South-East Pacific 102 to 104 mL−1, respectively. They play a fundamental Ocean gyre (hyper-oligotrophic), a transition region between role (Azam et al., 1983; Sherr and Sherr, 2000), in particular, the gyre and the coast of South America (moderately olig- in oligotrophic waters (Hagstrom¨ et al., 1988; Maran˜on´ et al., otrophic), and the Chile upwelling (eutrophic). The abun- 2001) where their small size associated to the reduced diffu- dance of phycoerythrin containing picocyanobacteria (PE sion boundary layer and large surface area per unit volume picocyanobacteria), autotrophic and heterotrophic eukary- are an advantage to acquire nutrients (Raven, 1998). The otes (classified into different size ranges), dinoflagellates, photosynthetic component of , i.e. Prochloro- and was determined by epifluorescence microscopy coccus and and picoeukaryotic after DAPI . Despite some apparent loss of cells algae, are important contributors to the microbial due to sample storage, distribution patterns were broadly of the euphotic zone in many marine environments (Mackey similar to those obtained by flow cytometry for PE pico- et al., 2002; Perez´ et al., 2006). Heterotrophic play cyanobacteria and . All populations reached a pivotal role in mediating organic flux to higher trophic a maximum in the Chile upwelling and a minimum near the levels in pelagic ecosystems (Azam et al., 1983; Fenchel, centre of the gyre. The maximum of PE pic- 1982; Hagstrom¨ et al., 1988). Among the heterotrophic pro- 3 −1 ocyanobacteria was 70 10 cell mL . Abundance of au- tists, ciliates and dinoflagellates are important grazers of pi- 3 totrophic eukaryotes and dinoflagellates reached 24.5 10 coplankton (Christaki et al., 2002). −1 and 20 cell mL , respectively. We observed a shift in the In the Pacific Ocean, picoplankton has been analyzed both size distribution of autotrophic eukaryotes from 2–5 µm in in the Equatorial region and the North gyre (e.g. Camp- eutrophic and mesotrophic regions to less than 2 µm in the bell et al., 1997; Mackey et al., 2002) but not in the South central region. The contribution of autotrophic eukaryotes to gyre. The latter is the most oligotrophic environment of the total eukaryotes was the lowest in the central gyre. Maxi- −1 world based on SeaWifs imagery which provides esti- mum concentration of ciliates (18 cell mL ) also occurred mates of average surface chlorophyll a concentrations down in the Chile upwelling, but, in contrast to the other groups, to 0.02 mg m−3 (Morel et al., 2007). The BIOSOPE (Bio- their abundance was very low in the HNLC zone and near geochemistry and Optics South Pacific Experiment) cruise the Marquesas Islands. Two key findings of this work that explored this region sailing from the Marquesas Islands to could not have been observed with other techniques are the the coast of Chile. Along this transect, a gradient in trophic high percentage of PE picocyanobacteria forming colonies in conditions was encountered, from hyper-oligotrophic (gyre) the HLNC region and the observation of numerous dinoflag- to very eutrophic waters (Chile upwelling). The present ellates with bright green autofluorescence. study relied on epifluorescence microscopy to assess the dis- tribution in this region of phycoerythrin containing pico- cyanobacteria (called PE picocyanobacteria throughout the Correspondence to: D. Vaulot paper), autotrophic and heterotrophic eukaryotes (in par- ([email protected]) ticular dinoflagellates and ciliates). In contrast to faster

Published by Copernicus Publications on behalf of the European Geosciences Union. 312 S. Masquelier and D. Vaulot: Micro-organisms in the South-East Pacific

STB1

STB3 STB4

STB6

STB8

GYR2 STB11 STB14

STB17

STB20

Fig. 1. Map of the BIOSOPE cruise track superimposed on a SeaWiFS ocean colour composite, dark purple indicating extremely low values (0.018 mg m−3) of total chlorophyll a. Figure modified from Claustre et al. (2007). Stations analyzed by DAPI staining are labelled.

Table 1. Concentrations of the different populations enumerated in the present study. Values are averages for the six depths sampled at each station.

Picocyanobacteria containing Total Autotrophic Heterotrophic Total Autotrophic Heterotrophic Green Total phycoerythrin eukaryotes eukaryotes eukaryotes dinoflagellates dinoflagellates dinoflagellates dinoflagellates ciliates Station Latitude-Longitude mL−1 mL−1 mL−1 mL−1 mL−1 mL−1 mL−1 mL−1 mL−1 MAR1 08◦23 S–141◦14 W 3486 1520 1292 228 105 56 48 4.6 <1.5 HLN1 09◦00 S–136◦51 W 2818 2312 1836 476 93 61 32 4.2 3 STB1 11◦44 S–134◦06 W 1612 1895 1165 730 111 62 50 4.5 1.5 STB3 15◦00 S–129◦55 W 413 1423 737 686 59 28 31 4.2 3.5 STB4 17◦13 S–127◦58 W 374 1267 736 531 57 26 32 7.0 1.5 STB6 20◦26 S–122◦54 W 6 1413 726 687 37 19 17 2.2 1.5 STB8 23◦32 S–117◦52 W 37 937 521 416 31 12 19 3.5 4.5 GYR2 25◦58 S–114◦00 W 46 806 541 265 43 21 22 3.5 1.5 STB11 27◦45 S–107◦16 W 34 1050 526 525 31 10 21 6.5 <1.5 STB14 30◦02 S–98◦23 W 142 1314 854 460 55 22 33 8.5 4.2 EGY2 31◦50 S–91◦27 W 1734 3083 2481 602 82 47 35 6.5 1.9 STB17 32◦23 S–86◦47 W 1104 2607 2086 521 94 46 48 12 5.2 STB20 33◦21 S–78◦06 W 10 726 1760 1195 566 92 44 48 6.7 3 UPW1 34◦01 S–73◦21 W 40 548 3396 2526 870 122 63 59 15 10 UPX2 34◦37 S–72◦27 W 18 548 14 088 12 211 1877 151 47 104 38 6.6

enumeration techniques such as flow cytometry, epifluores- tigated extended from the Marquesas Islands (South Pacific cence microscopy allows (1) to discriminate specific group of Tropical Waters; SPTW) to the coast of Chile, through the organisms such as dinoflagellates, (2) to recognize cell orga- Eastern South Pacific Central Waters (ESPCW) which in- nization such as colonies, and (3) to regroup organisms into clude the centre of the Pacific gyre (Claustre et al., 2008). size classes. We attempted to relate the distribution of the The transect can be divided into four contrasted trophic different types of organisms to oceanographic conditions. zones (from West to East): a High Nutrient Low Chlorophyll (HNLC) zone (mesotrophic) near the equator, the South-East Pacific gyre (hyper-oligotrophic) proper, the transition zone 2 Material and methods between the gyre and the coastal region (moderately olig- otrophic), and the Chile upwelling (very eutrophic). In the 2.1 Oceanographic context hyper oligotrophic zone, nitrate concentrations were nearly undetectable between the surface and 150–200 m and re- The BIOSOPE cruise took place on board the French NO mained very low (∼2.5 µM) below this depth (Fig. 2 in “l’Atalante” in the South-East Pacific Ocean from 26th Oc- Raimbault et al., 2007). Nitrate concentrations were higher tober to 11th December 2004 (Fig. 1). The transect inves- in the HNLC zone and maximum in the Chile upwelling

Biogeosciences, 5, 311–321, 2008 www.biogeosciences.net/5/311/2008/ S. Masquelier and D. Vaulot: Micro-organisms in the South-East Pacific 313

aa b5 µm c

Fig. 3. Heterotrophic (a), autotrophic (b), and green fluorescing a dinoflagellates (c) observed under blue light excitation (top) and UV light excitation (bottom). Pictures taken at stations STB3 (20 m), UPW and STB7 (5 m), respectively.

system equipped with 12 L Niskin bottles. In general, two samples were collected in the surface layer, three around the chlorophyll maximum and one below. Water was pre-filtered through a 200 µm mesh to remove , large phyto- , and particles before further filtrations. Water samples (100 mL) were fixed with glutaraldehyde (0.25% final concentration) and filtered through 0.8 µm pore c b size filters. This porosity was selected to avoid high densities of on the filter which would have rendered visuali- sation of the larger and less dense eukaryotes more difficult. Samples were stained with 4’6-diamidino-2-phenylindole (DAPI, 5 µg mL−1 final concentration) (Porter and Feig, 1980) and stored at –20◦C for a minimum of 12 months be- fore counting. Counts were performed with an Olympus BX51 epifluorescence microscope (Olympus Optical CO, Tokyo, Japan) equipped with a mercury light source and an x100 UVFL objective. Pictures of dinoflagellates were taken on board the ship on the freshly prepared slides using a BH2 Olympus microscope with an x40 objective and a Canon G5 digital camera. Pictures of PE containing picocyanobacte- 5 µm ria were taken in the laboratory on the BX51 Olympus mi- d croscope with a Spot RT-slider camera (Diagnostics Instru- ments, Sterling Heights, MI). Fig. 2. Pictures of single (a), and colonial PE picocyanobacteria (b- Prochlorococcus cannot be counted reliably by epifluores- d). Colony of more than 100 cells (b). Colony of 20–30 cells (c). cence microscopy because of their small size and rapidly Chain forming cells (d). Pictures taken under green light excitation fading fluorescence. Therefore, only isolated and colonial on samples of stations MAR1 at 80 m (a), MAR1 at 40 m (b), HNL1 PE picocyanobacteria (Fig. 2) were counted based on the at 60 m (c), and STB3 at 60 m (d). orange fluorescence of phycoerythrin excited under green light (530–550 nm). DAPI staining allowed us to discrimi- nate eukaryotic from prokaryotic organisms. Under UV light (Fig. 2 in Raimbault et al., 2007). Phosphate was apparently (360/420 nm), eukaryotic appeared as a separate never a limiting factor (Fig. 2 in Raimbault et al., 2007). blue , while for prokaryotes, no nucleus was visible and cells appeared uniformly stained. The red fluorescence 2.2 DAPI staining and epifluorescence microscopy of chlorophyll under blue light (490/515 nm) allowed us to discriminate autotrophic (photosynthetic) from heterotrophic Fifteen stations (Fig. 1 and Table 1) were sampled at six eukaryotes. However, it was not possible to distinguish depths with a conductivity-temperature-depth (CTD) rosette truly autotrophic organisms from organisms that had ingested www.biogeosciences.net/5/311/2008/ Biogeosciences, 5, 311–321, 2008 314 S. Masquelier and D. Vaulot: Micro-organisms in the South-East Pacific

) rately. Ciliates were discriminated by their shape, their size -1 (between 20 µm and 100 µm), and the presence of cilia and 200200000 000

(cell mL (cell a multiple nuclei. No distinction between different types of ciliates was attempted. Because of their low abundance, 50 fields per slide were counted for dinoflagellates and cil- 150150000 000 iates such that the minimum concentration detectable was 1.5 cell mL−1.

100100000 000 2.3 Data representation

5050000 000 Contour maps showing the distributions of the different pop- y = 3.06x ulations were drawn using the Ocean Data View software R2 = 0.96 (Schlitzer, 2003) with averaging VG gridding length-scales

0 of 100 for both X and Y. 0 2000020 000 40 40000 000 6060000 000

Unicellular picocyanobacteria PE by Unicellular PE picocyanobacteria by DAPI staining (cell mL -1 ) 3 Results )

-1 3.1 Comparison between microscopy and flow cytometry 1414000 000 b y = 2.36x R2 = 0.69 1212000 000 In order to validate our microscopy counts, we compared them to counts of Synechococcus cyanobacteria and photo- 1010000 000 synthetic eukaryotes done by flow cytometry (Grob et al.,

8000 2007) at the same stations (Fig. 4). There was a rela- 8 000 y = 1.5x tively good correlation between the two methods, such that R2 = 0.90 66000 000 global distribution trends were identical. However, slopes

44000 000 were significantly larger than one indicating that microscopy was underestimating the actual concentrations. For PE pic- 22000 000 ocyanobacteria (R2=0.96; n=80), abundance found by mi-

0 croscopy was 3 times lower than measured by flow cytome- 02 2000 000 4 4000 000 66000 000 8 8000000 Autotrophic (cell flow eukaryotesmL cytometry by try (Fig. 4a). For photosynthetic eukaryotes, the correlation Autotrophic eukaryotes by DAPI staining (cell mL -1 ) was moderate (R2=0.69; n=80) when all the data were con- sidered, although the slope was lower than for cyanobacteria − Fig. 4. Relationship between abundance (cell mL 1) measured by (Fig. 4b). When only data below 40-60 m were included, the flow cytometry (Grob et al., 2007) and estimated by DAPI counting correlation was significantly better (R2=0.90; n=56) and the for unicellular PE picocyanobacteria (a), and autotrophic eukary- slope less pronounced. otes (b). In panel (b) circles correspond to data from surface to 40–60 m depth depending on samples. Squares correspond to data 3.2 PE picocyanobacteria from 40–60 m depth to 300 m depth. (a) R2=0.96, n=80; (b) Solid 2 line takes into account all data (circles and squares); R =0.69 n=80. In surface, abundance of PE picocyanobacteria (Fig. 5a) Dashed line takes into account only squares; R2=0.90, n=56. reached a maximum (70 103 cell mL−1) near the coast of Chile (station UPW1) and a minimum (less than chlorophyll-containing cells. Ten fields and a minimum of 500 cell mL−1) in the middle of the South-East Pacific gyre. 100 cells were counted per slide. Eukaryotes were clas- Their abundance increased again near the Marquesas Is- sified according to three diameter ranges: (i) smaller than lands. In the vertical dimension, abundance decreased 2 µm, (ii) between 2 µm and 5 µm, (iii) larger than 5 µm. slightly down to circa 100 m and cells quickly disappeared Among eukaryotes larger than 5 µm, ciliates and dinoflagel- below (Fig. 5a). Interestingly, a large fraction of the PE lates were counted separately. Dinoflagellates were discrim- picocyanobacteria belonged to colonial forms in the vicinity inated by their shape, their size (between 5 µm and 100 µm), of the Marquesas Islands and in the HNLC zone (Fig. 5b). and the presence of a nucleus with condensed chromatin. In this region, this fraction could reach up to 50% near Autotrophic and heterotrophic dinoflagellates were discrim- the surface and 5 to 10% between 25 and 100 m, while it inated according to the red fluorescence of chlorophyll un- dropped below 5% almost everywhere else. Three types of der blue light of the former (Figs. 3a and b). Among het- colony could be observed (Fig. 2): (i) groups of 20–30 cells, erotrophic dinoflagellates, some were characterized by an in- (ii) groups of more than 100 cells, (iii) short chains. None tense green fluorescence under blue light (Fig. 3c), as re- of these forms seemed to be preferentially observed in any ported previously (Shapiro et al., 1989), and counted sepa- given region.

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(cell mL -1) (cell mL -1)

a a

(cell mL -1)

b b

Fig. 5. Abundance obtained by DAPI counting for unicellular PE picocyanobacteria (cell mL−1) (a), and percentage of unicellular PE picocyanobacteria in colony (b). Black dots correspond to sam- ples analysed. Contour plots generated with the software Ocean Data View.

3.3 Eukaryotes c

Maximum abundance of total eukaryotes (26 103 cell mL−1) occurred in the Chile upwelling near the surface (station Fig. 6. Abundance obtained by DAPI counting of total eukaryotes UPX2, 25 m) and minimum (276 cell mL−1) in the gyre at (cell mL−1) (a), autotrophic eukaryotes (cell mL−1) (b), and per- depth (station GYR2, 270 m) (Fig. 6a). In the surface layer, centage of heterotrophic eukaryotes in comparison with total eu- abundance was minimal in the center of the gyre and in- karyotes (c). Legend as in Fig. 5. creased both eastward and westward. The maxima of to- tal eukaryotes coincided roughly with the depth of chloro- phyll maximum (DCM, see Fig. 3 in Raimbault et al., gyre and the upwelling. Cells larger than 5 µm accounted for 2007). Below 200 m, concentrations were always lower than less than 10% of autotrophic eukaryotes everywhere along 1000 cell mL−1. The distributions of total eukaryotes and the transect, except near the Marquesas Islands where they autotrophic eukaryotes were very similar with a maximum contributed slightly more (Fig. 7c). in the Chile upwelling and a minimum in the surface of the The relative proportion of heterotrophic eukaryotes was gyre (Fig. 6b). These similar distributions were a mere con- the highest in the 0–50 m layer of the gyre (75–80%), while sequence of the fact that autotrophic eukaryotes were much in the DCM it dropped to 25% (Fig. 6c). In the DCM, cells more abundant than heterotrophic ones around the DCM smaller than 2 µm accounted for 28% (east of the gyre) to (Fig. 6c). The size distribution of autotrophic eukaryotes 40% (in the gyre) of heterotrophic eukaryotes (Fig. 8). The varied dramatically throughout the transect (Figs. 7 and 8): contribution of cells between 2 µm and 5 µm did not vary in the surface of the gyre, cells smaller than 2 µm accounted much (about 50%) while cells larger than 5 µm accounted for less than 10% while, they dominated (50–70%) in the for up to 14% of total heterotrophic eukaryotes in the HNLC DCM of the gyre as well as east of the gyre (Fig. 7a). In region and for about 10% elsewhere. the Chile upwelling (station UPX2, 25 m), they accounted In the 0–100 m layer, dinoflagellate abundance (Fig. 9a) for up to 80% of the total eukaryotes. In contrast, their con- increased towards the HNLC region (maximum observed: tribution was much lower in the HNLC region where larger 200 cell mL−1 at station STB1, 25 m) and the Chile up- eukaryotes between 2 µm and 5 µm accounted for 40% to welling, and decreased towards the gyre (minimum ob- 60% of the population (Fig. 7b). This size class was also served: 10 cell mL−1 at station GYR2, 270 m). In rela- dominant near the surface in the transition zone between the tive terms, autotrophic dinoflagellates dominated around the www.biogeosciences.net/5/311/2008/ Biogeosciences, 5, 311–321, 2008 316 S. Masquelier and D. Vaulot: Micro-organisms in the South-East Pacific

HNLC Gyre East of gyre Upwelling 100%

80%

60%

40%

Contributionto total 20% a

autotrophic/heterotrophic eukaryotes 0%

ro to te uto tero u Auto e A Auto e A H Hetero H Hetero

HNLC: MAR1, HLN1, STB1, STB3, STB4 > 5 µm Gyre: STB6, STB8, GYR2, STB11 > 2 µm and < 5 µm East of gyre: STB14, EGY2, STB17, STB20 < 2 µm Upwelling: UPW1, UPX2

Fig. 8. Contribution of the different size classes to the abundance of autotrophic (Auto) and heterotrophic (Hetero) eukaryotes at the b depth of chlorophyll maximum for the HNLC, Gyre, East of gyre and Chile upwelling regions.

At other stations, the maximum abundance of heterotrophic dinoflagellates was observed above the DCM, except in the upwelling (station UPX2) where the maximum was found below. At station EGY2 (east of gyre), the lowest concentra- tion of heterotrophic dinoflagellates (18 cell mL−1) occurred in the DCM. Green fluorescing dinoflagellates (Fig. 3c) accounted for c up to 50% of the heterotrophic dinoflagellates in surface east of the gyre and at depth in the Chile upwelling. They ac- counted for 5 to 25% of heterotrophic dinoflagellates in the Fig. 7. Fraction of autotrophic eukaryotes smaller than 2 µm (a), between 2 µm and 5 µm (b), and larger than 5 µm (c) in comparison HNLC zone and in surface in the Chile upwelling (Fig. 9d). −1 with the total eukaryotes. Legend as in Fig. 5. abundance reached a maximum (18 cell mL ) in the Chile upwelling (station UPW1, 40 m depth) and a min- imum in the HNLC region (Fig. 11). Abundance increased Marquesas Islands (up to 80% of total dinoflagellates, at sta- towards the Chile upwelling and decreased towards the gyre tion STB1, 80 m depth) and in the Chile upwelling (70% at as for most other groups. However, in contrast to most other station UPW1, 15 m depth) (Fig. 9b). The maximum of per- groups, ciliates also remained quite low towards the HNLC centage of autotrophic dinoflagellates (50%–80%) followed zone and the Marquesas Islands. Vertically, at many sta- the DCM except at station STB8 where the highest percent- tions, ciliate maxima corresponded to dinoflagellate minima age (50%) occurred at 70 m whereas the DCM was found (Fig. 10). much below at 170 m (Compare Fig. 9b in the present study and Fig. 3 in Raimbault et al., 2007). In the Chile upwelling, 4 Discussion the maximum of autotrophic dinoflagellates (50% at station UPX2 in surface and 70% at station UPW1 at 15 m) occurred Differences between abundances estimated by microscopy above the DCM. The percentage of autotrophic dinoflagel- vs. flow cytometry observed in this study could be due to sev- lates was the lowest (5%–25%) in the surface of the gyre and eral reasons. First, some cells smaller than 0.8 µm (e.g. some below 250 m. Synechococcus) could have passed through the 0.8 µm filter Heterotrophic dinoflagellates contribution ranged from used here. The loss of eukaryotic cells is however likely to be 20% to 95% of the total (Fig. 9c) and consisted mostly (75% negligible since the smallest known on average) of cells smaller than 15 µm in size (data not tauri has a size of 0.8 µm (Courties et al., 1994). Further- shown). Vertical profiles showed that maximum abundances more, according to Sherr et al. (2005), 16 % of Synechococ- of heterotrophic dinoflagellates followed the DCM only at cus and only 2% of cells may pass through a some stations in the gyre (STB3, STB6 and STB8, Fig. 10). 1 µm filter. This may explain why the slope in the Fig. 4 is

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(Cell mL -1)

a b

c d

Fig. 9. Total dinoflagellates (cell mL−1) (a), percentage of autotrophic dinoflagellates over total dinoflagellates (b), percentage of het- erotrophic over total dinoflagellates (c), and percentage of green dinoflagellates over total heterotrophic dinoflagellates (d). Legend as in Fig. 5. larger for PE picocyanobacteria than for eukaryotes. Second, ple fixation and conservation (Fig. 4). Therefore, colonial PE samples for microscopy were stored for more than a year picocyanobacteria would represent at least 15% (i.e. 33% of at −20◦C before counting while samples for flow cytome- 50%) of all PE picocyanobacteria. try were analyzed fresh on board. Gundersen et al. (1996) and Putland et al. (1999) found significant losses for bacteria The high proportion of colonial PE picocyanobacteria near and for unicellular cyanobacteria after one month and three the Marquesas Islands and in the HNLC region could be due months, respectively, of sample storage at −20 ◦C. Third, to a preference of colony-forming cyanobacteria for high nu- such storage conditions may cause a degradation of chloro- trient waters (Paerl, 2000). However, the fact that we ob- phyll and an underestimation of red fluorescing organisms served only 1% of colonial PE picocyanobacteria in the Chile (Chavez et al., 1990). Therefore, abundances of unicellular upwelling seems to indicate that other factors have to be PE picocyanobacteria and autotrophic eukaryotes may be un- taken into account. Some cyanobacteria encountered in ma- derestimated, while the proportion of heterotrophic eukary- rine systems form colonies (Graham and Wilcox, 2000) but otes could be higher than in the initial samples. In fact, we these are usually much larger than those we observed. It observed that organisms from surface samples had less in- is, for example, the case for Trichodesmium that has been tense chlorophyll fluorescence than those of deeper samples previously observed in the Equatorial Pacific (Capone et (as expected due to photoacclimation). Moreover, this fluo- al., 1997). Interestingly, unicellular PE picocyanobacteria rescence faded faster. Therefore the distinction between au- forming chains (cf. Fig. 2d) were isolated in culture from totrophic and heterotrophic organisms near the surface was the HNLC station at 30 m and 100 m depth (Le Gall et al., not always easy. This could explain the lower correlation and 2008) but the other morphotypes observed in the field were higher slopes between flow cytometry and epifluorescence not obtained. Since there was some evidence of nitrogen abundances for samples above 40–60 m (Fig. 4). fixation activity in this area (Raimbault and Garcia, 2007), it is tempting to hypothesize that these colonial PE pico- The low abundance of PE picocyanobacteria in the gyre cyanobacteria could be nitrogen-fixing. However, Raimbault and their higher abundance in the Chile upwelling, a region and Garcia (2007) showed that important nitrogen fixation rich in nutrients and characterized by mixed waters, is in was also observed in the center of the gyre and in the Chile agreement with many studies (for a review see Partensky et upwelling where very few colonies were observed. Further- al., 1999). Interestingly, up to 50% of the PE picocyanobac- more, small cyanobacteria having the capacity to fix nitrogen teria counted by microscopy appeared to be colonial near the do not seem to form colonies (Zehr et al., 2001). Their mor- Marquesas Islands and in the HNLC region (Figs. 2 and 5b). photype (spherical 3–10 µm cells) has been rarely observed Comparison with flow cytometry data showed that at least in our samples (data not shown). Alternatively, colony for- 33% of the PE picocyanobacteria were retained by the 0.8 mation could be an adaptation to the structure of the predator µm filter, assuming that no cells were lost because of sam- community in this region, such as the higher dinoflagellates www.biogeosciences.net/5/311/2008/ Biogeosciences, 5, 311–321, 2008 318 S. Masquelier and D. Vaulot: Micro-organisms in the South-East Pacific

Heterotrophic (cell mL -1 ) ton and to overestimate the contribution of macrophytoplank-

0 20 40 60 80 100120 140 1600 20 40 60 80 100120140 1600 20 40 60 80 100120140 160 ton. For example, they only took into account for the pi- 0 coplankton size group pigments characterizing cyanobacte- 50 ria and Chlorophyta. However, Prymnesiophyceae may also 100 contribute significantly to picoeukaryotic population (Moon- 150 Van Der Staay et al., 2001; Not et al., 2005). Indeed, Prym- 200

250 nesiophyceae cells characterized by two were MAR1 HLN1 STB1 30000 observed in our DAPI samples (data not shown). Conversely,

50 Ras et al. (2007) include pigments of dinoflagellates and di-

100 atoms in the microplankton size range (20–200 µm), while

150 many dinoflagellates and some smaller than 20 µm

200 (data not shown) have been detected along the South-East Pa- ◦ 250 cific transect, as observed previously along 110 W (Hardy et STB3 STB4 STB6 30000 al., 1996). Therefore, the contribution of microphytoplank- 50 ton could be overestimated. 100 During the BIOSOPE cruise, Gomez´ et al. (2007) found 150 −1

Depth (m) dinoflagellate abundance always lower than 1 cell mL , ex- 200 cept at station 20 where a bloom of dinoflagellates was ob- 250 ∼ −1 STB8 GYR2 STB11 served ( 4 cell mL between surface and 5 m depth), and 30000 at station UPW (∼2 cell mL−1). These counts from acid- 50 ified lugol’s fixed samples are much lower than ours (Ta- 100 ble 1). These differences could originate from differences 150

200 in the size of the dinoflagellates that were counted in these

250 two studies. We counted dinoflagellates which were between STB14 EGY2 STB17 30000 5 µm and 50 µm in diameter while Gomez´ et al. (2007)

50 only counted dinoflagellates larger than 15 µm. Hardy et

100 al. (1996) showed that dinoflagellates larger than 20 µm ac-

150 -1 Heterotrophic dinoflagellates (cell mL ) counted only for 10 to 30% of total dinoflagellates in the Pa- -1 200 Ciliates (cell mL ) cific gyre. In our samples (data not shown), the contribution 250 of dinoflagellates larger than 15 µm to total dinoflagellates STB20 UPW1 UPX2 300 in terms of abundances was below 1% near the Marquesas 0 5 10 15 200 5 10 15 200 5 10 15 20 -1 Ciliates (cell mL ) Islands, 1% in the upwelling zone, 2% in the HNLC zone and around the station EGY, and reached a maximum of 3% Fig. 10. Vertical profiles of concentration (cell mL−1) of total het- at station ST20 where a bloom was observed by Gomez´ et erotrophic dinoflagellates (solid line) and ciliates (dotted line). Stars al. (2007). indicate the depth of chlorophyll maximum. Globally, the abundance of dinoflagellates (Fig. 9) de- creased towards the hyper-oligotrophic zone and increased to ciliates ratio. Indeed, cells forming colonies could take ad- towards the mesotrophic and eutrophic zones. This is in vantage of the positive aspects of increased size, in particular agreement with Leterme et al. (2006) who showed that the lower grazing pressure, without paying the full cost of de- dinoflagellate abundances increased with trophic status in the creased metabolism and reduced growth which is associated NE Atlantic Ocean. The observed increase in heterotrophic with large individual cell size (Nielsen, 2006). In the light of dinoflagellates contribution with depth is coherent with pre- our observations, it could be interesting to extend counts of vious observations in the Equatorial Pacific (Chavez et al., colonial picocyanobacteria to other oceanic regions in order 1990). Heterotrophic dinoflagellates were always much to better understand how this fraction varies with oceano- more abundant than ciliates as shown previously in the Sar- graphic conditions. gasso Sea (Lessard and Murrell, 1996) and the North-East The present study is consistent with estimates by Ras et Equatorial Pacific (Yang et al., 2004). Although it is gener- al. (2007) based on HPLC pigment data and assumptions ally admitted that heterotrophic nanoflagellates are the ma- concerning the size range of different taxonomic groups jor grazers of picoplankton (Mackey et al., 2002; Sato et al., (Claustre, 1994; Vidussi et al., 2001). They found that the 2007), predation by heterotrophic dinoflagellates could also contribution of picophytoplankton (in terms of percentage be important (Sanders et al., 2000; Sherr et al., 1991). of total chlorophyll a) was the highest in the gyre itself Green fluorescing dinoflagellates were initially observed and east of gyre, while dominated in the by Shapiro et al. (1989) in the North-West Atlantic, but lit- HNLC zone and the Chile upwelling. However, their method tle reported since then. Recently, Tang and Dobbs (2007) tends to underestimate the contribution of picophytoplank- showed that green autofluorescence is a common feature

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of dinoflagellates, diatoms, , cyanobacteria and (Cell mL -1) . They observed that this green autofluores- cence is stronger in fixed cells, not stable over time, and that its intensity varies with organisms (the strongest signal is observed for dinoflagellates). Shapiro et al. (1989) found that green fluorescing dinoflagellates could contribute from 4 to 100% to heterotrophic dinoflagellates while Chavez et al. (1990) found that in the Equatorial Pacific, 32% of het- erotrophic dinoflagellates on average produced bright green fluorescence. The data reported here (maximal concentra- tions in excess of 60 cell mL−1 and maximum contribution −1 up to 50 %, Fig. 9) are in agreement with these previous stud- Fig. 11. Abundance of ciliates (cell mL ). Legend as in Fig. 5. ies. The origin of this bright green fluorescence (Fig. 3c) still remains intriguing. Shapiro et al. (1989) hypothesized that Sherr et al., 1991) and account in general from 50 to 95% it could be due to a flavoprotein. The isolation by Fujita et of total ciliates in a variety of marine ecosystems (Beers et al. (2005) of a flavoprotein from the green-fluorescing flag- al., 1980; Montagnes et al., 1988; Yang et al., 2004). How- ellum of the brown alga Scytosiphon lomentaria lends sup- ever, in our study, we observed very few nanociliates as the port to this hypothesis. Kim et al. (2004) showed that the size of the majority of ciliates felled into a 50–100 µm range infection of the thecate dinoflagellate Gonyaulax spinifera (data not shown). That could be explained by the fixation by Amoebophrya, a parasitic dinoflagellate, induces a bright method used in our study: Leakey et al. (1994) demonstrated green autofluorescence in infected cells. This fluorescence that the use of glutaraldehyde as fixative could lead to a is, however, much more localised than in the green dinoflag- loss of cells as high as 70% among aloricate ciliates rela- ellates observed in our samples (Fig. 3c). Another attractive tive to lugol’s iodine while tintinnid numbers did not vary possibility could be the presence of a cytoplasmic green flu- significantly between fixative treatments. However, Dolan orescing protein (GFP, Wilson and Hastings, 1998). and Marrase´ (1995), observed only 8% of nanociliates in the Ciliate abundances reported here (Table 1) are comparable Catalan Sea in June 1993 while lugol’s iodine was used as to those reported from other similar marine systems ranging fixative. from oligotrophic to eutrophic (Beers et al., 1980; Leakey In conclusion, although assessing the abundance of the dif- et al., 1996; Lessard and Murrell, 1996; Yang et al., 2004). ferent microbial groups by DAPI microscopy is slow and Focusing only on tintinnid ciliates, Dolan et al. (2007) ob- labour-intensive and despite some cell loss following long- served during the BIOSOPE cruise much lower concentra- term sample storage, the present data set highlights some tions ranging from 0.002 and 0.04 cell mL−1 between 5 and characteristics of the microbial community in the South-East 300 m. However, tintinnids generally account only for 5– Pacific Ocean that have escaped more rapid techniques such 10% of all ciliates (Dolan and Marrase,´ 1995). Comparing as flow cytometry. This includes in particular the importance our data with values from Table 2 of Dolan et al. (2007) re- of colonial PE containing picocyanobacteria in the HNLC sults in a proportion of tintinnids (0.05%) smaller, for ex- area and the large contribution of green fluorescing dinoflag- ample, than in the Catalan Sea (Dolan and Marrase,´ 1995). ellates in some regions, such as between the gyre and the However, maxima and minima of tintinnid and total ciliates coast of South America. occurred simultaneously, in the upwelling and in the gyre, respectively. Acknowledgements. D. Tailliez and C. Bournot are warmly The distribution pattern of ciliates (Fig. 11) agrees with thanked for their efficient help in CTD rosette management and previous observations in the North Western Indian Ocean data processing. D. Marie, M. Viprey and L. Garczarek are ac- (Leakey et al., 1996) where the lowest abundances were ob- knowledged for their help during the cruise. This is a contribution served in oligotrophic waters and the highest in the most pro- to the BIOSOPE project (LEFE-CYBER program). This research ductive waters. The different patterns of vertical distribution was funded by the Centre National de la Recherche Scientifique of ciliates observed in the present study could be explained (CNRS), the Institut des Sciences de l’Univers (INSU), the by the fact that no distinction has been made between the Centre National d’Etudes Spatiales (CNES), the European Space Agency (ESA), the National Aeronautics and Space Administration different types of ciliates (mixotrophic and heterotrophic cili- (NASA) and the Natural Sciences and Engineering Research ates). In the Catalan Sea, the distribution of heterotrophic cil- Council of Canada (NSERC). Additional funds were provided iate is closely related to the DCM while mixotrophic ciliates by the ANR Biodiversity project PICOFUNPAC. S. Masquelier display a more complicated vertical pattern and their distri- was supported by a doctoral fellowship (BFR05/027) from the bution may vary from system to system (Dolan and Marrase,´ Ministere` de la Culture, de l’Enseignement Superieur´ et de la 1995). Recherche of Luxembourg. Nano-ciliates (<20 µm) have been identified as potentially important grazers of picoplankton (Sherr and Sherr, 1987; Edited by: M. Dai www.biogeosciences.net/5/311/2008/ Biogeosciences, 5, 311–321, 2008 320 S. Masquelier and D. Vaulot: Micro-organisms in the South-East Pacific

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